Recent developments in the field of DNA vaccination using recombinant viral vectors have created the need for large scale manufacturing of clinical grade material. Processes are needed to be able to support the less and least developed world with sufficient amounts of recombinant adeno-based vaccines to fight, e.g., the Tuberculosis and Malaria problem in the world. An evaluation of the birth cohort shows that more than 150,000,000 births are expected for the less and least developed world in 2010-2015. Based on this birth cohort, the projected annual demand for a vaccine could reach approximately 1.5×1019 virus particles (VP) on a yearly basis.
Several processes for production of adenoviruses have been described. These processes use adherent cell cultures in roller bottles, cell factories (Nunclon from Nunc or CellStack from Corning), or Cell Cubes (Corning). Production processes on adherent cell cultures cannot fulfill the worldwide demand for adeno-based vaccines. Therefore, the cells used in the adherent process are adapted to suspension cultures (e.g., HEK293 and PER.C6® cell lines). With the use of suspension cultures it is possible to scale-up production processes to large-scale bioreactors. Suspension cell cultures for adenovirus production are routinely achieved between 3 to 20 L scale and successful scale-up has been reported up to 100 L (Kamen et al., 2004), and 250 L (Xie et al., 2003). Experiments are reported in which scaling up to 10,000 L is anticipated (Xie et al., 2003).
However, a major disadvantage of scaling up to 10,000 L is the high capital investment (CAPEX), which is needed to design and build a 10,000 L bioreactor facility. Furthermore, the CAPEX commitment of building a 10,000 L facility, under BSL 2 conditions, must be realized before even knowing if the product will be successful (Phase IV and beyond). The total investment cost for a 10,000 L bioreactor plant is reported between 225,000,000 and 320,000,000 (Estape et al., 2006). Therefore, preparation at lower scale, e.g., in 1000 L or smaller bioreactors, would be desirable.
With the use of currently existing processes, more than 150 batches at 1000 L scale a year must be produced in order to reach the target of 1.5×1019 VP/year. Therefore, a need exists to improve systems for adenovirus production, to improve yields of adenovirus particles in order to fulfill the world-wide demand of adenovirus vaccines, preferably at non-prohibitive costs.
One of the issues encountered in adenovirus production optimization is the so-called “cell density effect.” In batch-mode operation, several references suggest the existence of an optimal cell density at infection for adenovirus production. The optimum lies between 0.5−1×106 cells/mL (Maranga et al., 2005; Kamen et al., 2004). It was shown for adenovirus (Ad5) production in a batch stirred tank bioreactor that the virus productivity per cell remains constant up to around 0.9×106 cells/mL, but drops abruptly at around 1×106 cells/mL (Altaras et al., 2005). Beyond 2×106 cells/mL, no infectious particles were detectable. The breakpoint related to specific production drop with cell densities at infection is medium dependent. No available commercial medium to date has shown potential to support high yields of virus particles, while maintaining the specific production optimal at cell densities beyond 1×106 cells/mL (Kamen et al., 2004). The reasons for this drop are not known yet but might be due to limited nutrient availability for virus production, or due to high metabolites concentrations that are inhibitory for virus production.
Fed-Batch operations, like addition of glucose, glutamine and amino acids allowed infections at cell densities up to 2×106 cells/mL. However, the productivities attained at high-cell densities were lower than those obtained with infection at cell densities of 1×106 cells/mL (Kamen et al., 2004).
In perfusion processes, the cells are retained in the bioreactor by hollow fibers, spin filters or acoustic separators while culture medium is perfused through the bioreactor. In these processes cell densities of >100×106 cells/mL can sometimes be reached (e.g., Yallop et al., 2005).
Infected perfusion cells showed premature cell loss during perfusion with a hollow fiber system. This might be related to their higher shear sensitivity due to the viral infection (Cortin et al., 2004). The hydro-dynamical stresses induced in the tubing, the hollow fibers, or the peristaltic pump on more fragile, infected cells was most likely the cause for this phenomenon. Since infected cells are more fragile, particularly the acoustic separator (Henry et al., 2004) has been suggested to be desirable if the perfusion is to be maintained throughout the infection phase. However, infections performed in perfusion mode could only be maintained for cell densities up to 3×106 cells/mL with a perfusion rate of 2 vol/day. Infection at a cell density of 6×106 cells/mL led to a fivefold reduction in specific productivity (Henry et al., 2004).
Despite the reported cell density effect by others, one report (Yuk et. al., 2004) described successful perfusion cultures of human tumor cells as a production platform for oncolytic adenoviral vectors. That report described a high-cell-density perfusion process using alternating tangential flow (ATF) technology. At an average viable cell density at an infection of 9×106 HeLaS3 cells/mL, an average viral titer of about 4×1011 VP/mL was observed. The tumor cells used in that report are not preferred as production cells, since use of tumor cells may pose safety risks when the produced adenovirus particles are to be administered to humans. The recombinant adenovirus in that report was based on Ad5. Such adenoviruses have limited possibilities for use as vaccines since a majority of the human population has pre-existing neutralizing antibodies against Ad5, and recombinant adenoviruses from other serotypes are therefore more suitable for use as vaccines (see, e.g., WO 00/70071). In particular, recombinant adenoviruses from subgroup B, such as Ad35, are especially advantageous for use as vaccines (WO 00/70071).
Limited information, if any, is available for the large scale production of recombinant adenoviruses from other serotypes than Ad5, in particular for the advantageous serotype 35. Some differences between Ad35 and Ad5 have been described with respect to purification thereof using anion exchange (e.g., WO 2005/080556). The somewhat different physical properties of recombinant adenoviruses of different serotypes may give rise to differences in production processes or under certain conditions. Such potential differences may especially be important at industrial scale, where even seemingly small differences at small scale may have large economic consequences on the scale envisaged for production of the annual world-wide demand. For instance, it is hitherto unknown whether the reported cell density effect for Ad5 will be similar for other serotypes. Therefore, in order to fulfill the world-wide demand of rAd35 vaccines, a need exists to improve systems for recombinant adenovirus serotype 35 (rAd35) production.